SRC1 deficiency in hypothalamic arcuate nucleus increases appetite and body weight

in Journal of Molecular Endocrinology
Correspondence should be addressed to G Ning or Q Ma: gning@sibs.ac.cn or qinyunma@126.com
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Appetite is tightly controlled by neural and hormonal signals in animals. In general, steroid receptor coactivator 1 (SRC1) enhances steroid hormone signalling in energy balance and serves as a common coactivator of several steroid receptors, such as oestrogen and glucocorticoid receptors. However, the key roles of SRC1 in energy balance remain largely unknown. We first confirmed that SRC1 is abundantly expressed in the hypothalamic arcuate nucleus (ARC), which is a critical centre for regulating feeding and energy balance; it is further co-localised with agouti-related protein and proopiomelanocortin neurons in the arcuate nucleus. Interestingly, local SRC1 expression changes with the transition between sufficiency and deficiency of food supply. To identify its direct role in appetite regulation, we repressed SRC1 expression in the hypothalamic ARC using lentivirus shRNA and found that SRC1 deficiency significantly promoted food intake and body weight gain, particularly in mice fed with a high-fat diet. We also found the activation of the AMP-activated protein kinase (AMPK) signalling pathway due to SRC1 deficiency. Thus, our results suggest that SRC1 in the ARC regulates appetite and body weight and that AMPK signalling is involved in this process. We believe that our study results have important implications for recognising the overlapping and integrating effects of several steroid hormones/receptors on accurate appetite regulation in future studies.

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  • Supplementary Figure S1 Distribution of SRC1 in the hypothalamic arcuate nucleus of mice. (A) A representative immunofluorescent staining of SRC1 (red) in coronal hypothalamic sections from AgRP-GFP mice. AgRP neurons are identified by GFP (green) fluorescence. (B) A representative immunofluorescent staining of SRC1 (red) in POMC-GFP mice. POMC neurons are also identified by GFP (green) fluorescence. All the mice were 8 weeks old and males and received a normal chow diet. Twelve sections from three of both types of mice were assessed. All images were generated by laser scanning confocal microscopy. 3V, Third ventricle. Scale bars (A, B), 100 μm.

 

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    Distribution of SRC1 in mouse hypothalamic nucleus. (A) A representative immunofluorescent staining for SRC1 (green) in mice. 10 sections from three mice, all images were generated by fluorescent microscope. 3V, third ventricle. Scale bar, 100 μm. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0075.

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    Expression of hypothalamic SRC1 in different nutritional conditions. (A) Hypothalamic Src1 expression was reduced by 24 h of fasting and partly restored by 6 h of refeeding. (B) The hypothalamic Src1 expression in mice fed with an HFD for 6 months was reduced compared with that in mice fed with NCD. mRNA was measured by quantitative real-time PCR and relative to 36b4. Data are presented as mean ± s.e.m. and were analysed using one-way ANOVA (A) and Student’s t-test (B). *P < 0.05, ***P < 0.001, n = 10 (A) or 8 (B) mice per group. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0075.

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    Construction of the Src1 shRNA lentivirus and efficiency of SRC1 in the ARC and GT1-7 cell line. (A and B) Lentivirus-mediated Src1 knockdown in the GT1-7 cell line. Src1 mRNA was measured by quantitative real-time PCR and relative to 36b4 (A). The SRC1 protein level was tested by western blotting, and β-actin levels were shown as the loading control (B). MOI = 60 pfu number/cell, n = 4 per group. (C and D) The hypothalamic Src1 (C), Src2 and Src3 (D) levels after bilateral ARC injection of the Src1 lentivirus (shRNA-SRC) compared with those after injection of shRNA-GFP examined by real-time PCR (n = 11–16 per group). (E, F, H and I) SRC1 and GFP staining in the ARC of mice receiving unilateral ARC injections in both NCD (E and F) and HFD (H and I). The mice were injected with shRNA-GFP into one side of the ARC and shRNA-SRC into the other side. Brain sections across the ARC were stained for GFP (E and H) or SRC1 (F and I) antibodies and observed by fluorescence microscopy. (G and J) Immunostaining quantification of SRC1-positive cells in the ARC (n = 3 per group, 8–10 sections per sample). 3V, third ventricle. Scale bar, 100 μm. Data are presented as mean ± s.e.m. and were analysed using Student’s t-test and two-way ANOVA (C). *P < 0.05, ***P < 0.001. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0075.

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    The ARC SRC1 knockdown regulates food intake. (A, B, C and D) Daily food consumption and accumulative food intake between the shRNA-SRC and shRNA-GFP mice that were fed with NCD (A and B) and HFD (C and D). Data are presented as mean ± s.e.m. and were analysed using Student’s t-test. *P < 0.05, **P < 0.01, n = 7–11 per group. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0075.

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    The ARC SRC1 knockdown regulates body weight but not via the mechanisms of energy expenditure or glucose production. (A and B) Changes in the body weight of shRNA-SRC and shRNA-GFP mice that were fed with NCD (A) and HFD (B). The change in body weight was based on the body weights on the first day after injection. (C) The weight of the white fat pads from shRNA-SRC and shRNA-GFP mice that were fed with HFD. (D and E) Oxygen consumption (VO2) (D) and carbon dioxide production (VCO2) (E) analysed by indirect calorimetry in shRNA-SRC and shRNA-GFP mice that were fed with HFD presented over three consecutive 12-h dark and two 12-h light cycles. (F) The Ucp1 and Pgc1α expression in the brown adipose tissue of shRNA-SRC and shRNA-GFP mice fed with HFD was measured by quantitative PCR and relative to 36b4. (G) Blood glucose levels of shRNA-SRC and shRNA-GFP mice fed with HFD after fasting overnight. (H and I) The G6pase (H) and Pepck (I) mRNA expression in the liver of shRNA-SRC and shRNA-GFP mice measured by quantitative real-time PCR. eWAT, epididymal adipose tissue; iWAT, inguinal adipose tissue; tWAT, total adipose tissue. Data are presented as mean ± s.e.m. and were analysed using Student’s t-test. *P < 0.05, **P < 0.01, n = 7–11 per group. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0075.

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    SRC1 knockdown in the ARC reduces ERα but activates the hypothalamic AMPK signalling pathway. (A and B) Serum leptin levels (A) and insulin levels (B) of shRNA-SRC and shRNA-GFP mice fed with HFD. (C) The mRNA expression of Erα in the hypothalamus. (D) ERα staining in the ARC of mice that received unilateral ARC injections. The mice were injected with shRNA-GFP into one side of the ARC and shRNA-SRC into the other side. The images were generated by fluorescence microscopy; nine sections from three mice were imaged, and one representative image was chosen. 3V, third ventricle. Scale bar, 100 μm. (E and F) AMPK phosphorylation and protein expression of the hypothalamus of shRNA-SRC or shRNA-GFP mice fed with HFD. Protein levels were measured by western blotting, and β-actin levels are shown as the loading control. The phosphorylated AMPK level of the hypothalamus (E) was quantified using Image J relative to β-actin and t-AMPK (F). (G and H) Hypothalamic phosphorylated S6K, S6, and β-actin protein levels of shRNA-SRC mice and shRNA-GFP mice with HFD were measured by western blotting, β-actin levels are shown as the loading control (G), The phosphorylated S6K and S6 level was quantified using Image J relative to β-actin (H). Data are presented as mean ± s.e.m. and were analysed using Student’s t-test. *P < 0.05, **P < 0.01, n = 13 per group (A, B and D), 4–5 per group (F and H). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0075.

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    Schematic representation of the role of SRC1 in the ARC. SRC1 in the ARC could be regulated by both hormone signals and nutrient signals. After interacting with some NRs, SRC1 suppressed AMPK phosphorylation and protein expression, thus modulating food intake. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0075.

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